1

Comparison of Salmonella enterica serovars Typhi and Typhimurium reveals typhoidal-1

specific responses to bile 2

3

Rebecca Johnson1, Matt Ravenhall

2, Derek Pickard

3, Gordon Dougan

3, Alexander Byrne

1*, 4

Gad Frankel1# 5

6

Running title: Salmonella Typhi bile responses 7

1MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, 8

Imperial College London, London, United Kingdom 9

2Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical 10

Medicine, London, United Kingdom 11

3Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, 12

United Kingdom 13

14

* Present address: Animal and Plant Health Agency, Weybridge, United Kingdom 15

16

#Corresponding author: Gad Frankel, g.frankel@imperial.ac.uk 17

IAI Accepted Manuscript Posted Online 11 December 2017Infect. Immun. doi:10.1128/IAI.00490-17Copyright © 2017 Johnson et al.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

2

ABSTRACT 18

Salmonella enterica serovars Typhi and Typhimurium cause typhoid fever and gastroenteritis 19

respectively. A unique feature of typhoid infection is asymptomatic carriage within the 20

gallbladder, which is linked with S. Typhi transmission. Despite this, S. Typhi responses to 21

bile have been poorly studied. RNA-Seq of S. Typhi Ty2 and a clinical S. Typhi isolate 22

belonging to the globally dominant H58 lineage (129-0238), as well as S. Typhimurium 23

14028, revealed that 249, 389 and 453 genes respectively were differentially expressed in the 24

presence of 3% bile compared to control cultures lacking bile. fad genes, the actP-acs 25

operon, and putative sialic acid uptake and metabolism genes (t1787-t1790) were upregulated 26

in all strains following bile exposure, which may represent adaptation to the small intestine 27

environment. Genes within the Salmonella pathogenicity island 1 (SPI-1), encoding a type 28

IIII secretion system (T3SS), and motility genes were significantly upregulated in both S. 29

Typhi strains in bile, but downregulated in S. Typhimurium. Western blots of the SPI-1 30

proteins SipC, SipD, SopB and SopE validated the gene expression data. Consistent with this, 31

bile significantly increased S. Typhi HeLa cell invasion whilst S. Typhimurium invasion was 32

significantly repressed. Protein stability assays demonstrated that in S. Typhi the half-life of 33

HilD, the dominant regulator of SPI-1, is three times longer in the presence of bile; this 34

increase in stability was independent of the acetyltransferase Pat. Overall, we found that S. 35

Typhi exhibits a specific response to bile, especially with regards to virulence gene 36

expression, which could impact pathogenesis and transmission. 37

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

3

INTRODUCTION 38

In humans, the outcome of infection with Salmonella enterica primarily depends on the 39

infecting serovar; whilst non-typhoidal, broad host range serovars such as Salmonella 40

enterica serovar Typhimurium (S. Typhimurium) cause self-limiting gastroenteritis, infection 41

with human-restricted typhoidal serovars, such as Salmonella enterica serovar Typhi (S. 42

Typhi) result in typhoid fever (1). The virulence of both serovars depends on the activity of 43

two type III secretion systems (T3SS) carried on Salmonella pathogenicity islands 1 and 2 44

(SPI-1 and SPI-2), which secrete a pool of over 40 effectors to subvert host cell processes 45

resulting in invasion, immune evasion, and intracellular growth (2). The SPI-1 T3SS is active 46

when Salmonella are extracellular, and its activity permits Salmonella invasion of non-47

phagocytic cells and also promotes early adaptation to the intracellular environment (2). 48

Expression of the SPI-1 T3SS and its associated genes (several of which are encoded outside 49

of the SPI-1 pathogenicity island) is controlled by a hierarchy of regulators (HilD, HilA, 50

HilC, RtsA, InvF). These regulators are controlled by a variety of factors including two-51

component systems, RNA binding proteins, and global regulators, which respond to a range 52

of environmental stimuli (3, 4). 53

Typhoid is an acute illness characterized by high fever, malaise and abdominal pain (5). S. 54

Typhi causes systemic infection during which the pathogen colonises the intestine and 55

mesenteric lymph nodes, the liver, spleen, bone marrow and gallbladder (5). It is estimated 56

that there are more than 20 million typhoid fever cases per year, resulting in more than 57

200,000 deaths (6). Although with adequate treatment most patients recover from the acute 58

phase of S. Typhi infection, S. Typhi can persist asymptomatically within the gallbladder 59

following clinical recovery (7). Overall, 10% of those infected will carry S. Typhi within 60

their gallbladder for up to three months, whilst 1-3% will continue to harbour S. Typhi for 61

longer than one year (5, 8). Given the host-restriction of S. Typhi, chronic gallbladder 62

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

4

carriage represents a key environmental reservoir of S. Typhi bacteria, enabling typhoid 63

transmission (7, 9). 64

Although the exact mechanism(s) by which S. Typhi persists within the gallbladder are 65

debated (7), it certainty encounters high bile concentrations during carriage, as the 66

gallbladder is where bile is stored and concentrated prior to secretion into the small intestine, 67

where it plays a role in the emulsification and absorption of fats (10). In part due to its 68

detergent activity, bile is also a potent antimicrobial agent (10, 11). However enteric 69

pathogens – including Salmonella – are intrinsically resistant to bile (12), and instead often 70

utilise bile as a means to regulate gene expression and virulence (10, 13). In S. Typhimurium, 71

expression of the SPI-1 and motility genes are repressed by bile exposure, resulting in a 72

significant repression of epithelial cell invasion (14, 15). 73

Despite the importance of asymptomatic carriage, the behaviour of S. Typhi within bile 74

remains poorly understood (7). As the transcriptomic responses of S. Typhimurium to bile 75

under various conditions have been well characterised (15–18), the behaviour of S. 76

Typhimurium has become an accepted model as to how Salmonella in general behaves in bile 77

(11, 19). However a study comparing changes in protein expression by 2D gel electrophoresis 78

within S. Typhimurium and S. Typhi following exposure to 3% bile found there was “little 79

overlap apparent between proteins affected by bile in S. Typhi and in S. Typhimurium” (12), 80

suggesting that the response to bile between these serovars differs. Furthermore, a study 81

comparing the genomes of S. Typhimurium LT2 to S. Typhi CT18 revealed that less than 82

90% of genes are shared between the two strains, with over 600 genes present in CT18 not 83

found in LT2 (20); therefore S. Typhimurium cannot be used to model regulation of S. Typhi 84

specific genes, which include key virulence factors such as the Vi antigen, and the CdtB and 85

HlyE/ClyA toxins (20). 86

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

5

The need to better understand S. Typhi infection has been intensified by the recent spread of 87

haplotype 58 (H58), also known as 4.3.1 (21, 22). Following its emergence around 30 years 88

ago, S. Typhi strains belonging to haplotype H58 have clonally expanded worldwide to 89

become the dominant cause of multi-drug resistant (MDR) typhoid within endemic regions 90

(21). As yet, the reasons underlying the relative success of H58 strains remain unknown. 91

The aim of this study was to compare global bile responses between S. Typhi and S. 92

Typhimurium isolates, which in turn might explain differences in pathogenesis and reveal 93

processes important for the carrier state. 94 on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

6

RESULTS 95

Bile exposure alters global gene expression in Salmonella 96

We performed RNA-Seq on S. Typhimurium 14028, S. Typhi Ty2 and a clinical S. Typhi 97

H58 isolate (129-0238) grown in LB to late-exponential phase in the presence or absence of 98

3% bile. Given the extensive description of S. Typhimurium behaviour in bile (14, 15), S. 99

Typhimurium 14028 was considered as a control. 3% ox-bile was chosen for these studies as 100

this concentration robustly affects gene expression in S. Typhimurium (14, 15, 23), but does 101

not affect growth of the investigated Salmonella strains (Figure S1). Overall following 102

growth in bile, 249 and 389 genes were differentially expressed in S. Typhi Ty2 (182 103

upregulated; 67 downregulated) and 129-2038 (223 upregulated; 166 downregulated) (Figure 104

1) respectively, while 453 genes were differentially regulated in S. Typhimurium 14028 (293 105

upregulated; 179 downregulated) (Figure 1). 106

GO enrichment and KEGG pathway analysis on the pools of upregulated and downregulated 107

genes revealed broad differences between S. Typhi and S. Typhimurium (Figure 1). While S. 108

Typhimurium upregulated metabolic processes and downregulated processes linked with 109

pathogenicity, including T3SS, flagella and chemotaxis (motility), in line with previous 110

findings (14, 15, 17), both S. Typhi Ty2 and 129-0238 upregulated these processes, whilst 111

downregulating various metabolic pathways (Figure 1). KEGG pathway analysis also 112

revealed that fatty acid degradation (represented by the GO term ‘Fatty acid beta-oxidation’) 113

and tyrosine metabolism were upregulated in all isolates, implicating these processes in 114

general Salmonella response to bile. 115

Similarities in the response to bile between S. Typhi and S. Typhimurium 116

The overlap in genes either downregulated or upregulated in bile between all strains was 117

small; only one gene (pagP), a PhoP-PhoQ regulated gene involved in modifying lipid A 118

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

7

(24), was downregulated in all strains (Figure 2). Twenty genes were upregulated in all 119

isolates in response to bile (Figure 2) (Table 1), representing genes involved in tyrosine 120

metabolism, sialic acid uptake and utilisation (t1787-1790) (25), and in the production of 121

acetyl-CoA from acetate (actP-acs) and fatty acids (fad genes). Of the upregulated genes, 122

expression of acs and fadE was validated by RT-qPCR (Table 2). Upregulation of sialic acid 123

and acetate metabolic pathways may reflect adaptation to the small intestine, where these 124

metabolites are abundant (26), whilst upregulation of fad genes are consistent with the ability 125

of Salmonella to utilise phospholipids present in bile as a carbon/energy source (27). 126

Interestingly the fatty acid transporter fadL, was strongly upregulated in S. Typhimurium, but 127

was not upregulated in either S. Typhi Ty2 or 129-0238, suggesting that S. Typhi may 128

possess additional fatty acid transporters. 129

Genes implicated in stress responses were also upregulated in bile. All isolates upregulated 130

msrA, a sulfoxide reductase upregulated in response to oxidative stress, which is required for 131

growth within macrophages and for full virulence of S. Typhimurium in vivo (28). S. 132

Typhimurium 14028 and S. Typhi 129-0238 also activated RpoS-mediated stress responses, 133

with upregulation of otsAB, spoVR, yeaG, katE, sodC, poxB, ecnB, and osmY, in line with 134

previous findings (17, 29, 30). However, upregulation of these stress-linked genes was not 135

observed in S. Typhi Ty2, which is likely due to a frameshift mutation within rpoS within this 136

strain (31). 137

Differences in the response to bile between S. Typhi and S. Typhimurium 138

Of special interest are genes that are regulated differently in response to bile between S. 139

Typhi and S. Typhimurium. The identification of such genes was achieved by determining 140

genes downregulated in S. Typhimurium in bile, but upregulated in S. Typhi and vice versa. 141

Of the 75 genes upregulated in both S. Typhi Ty2 and 129-0238 (Figure 2), the majority 142

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

8

(54/75) were significantly downregulated in S. Typhimurium (Table 3). As indicated by the 143

GO and KEGG pathway analyses (Figure 1), genes regulated in this manner predominantly 144

encode proteins associated with the SPI-1 T3SS or motility. To validate these findings, 145

expression of the SPI-1 associated genes hilD, hilA, prgH, and sopB, in addition to the 146

flagella associated genes flhD and flgA was confirmed by RT-qPCR (Table 2). 147

Additional genes upregulated in S. Typhi and downregulated in S. Typhimurium include lpxR 148

(t1208/STM14_1612), a lipid A modifying protein that modulates the ability of lipid A to 149

stimulate TLR4 (32) and promotes Salmonella growth inside macrophages (33), and 150

srfA/srfB, virulence factors expressed under SPI-1 inducing conditions (34) and reported to 151

modulate inflammatory signalling (35). Additionally, several hypothetical proteins – t0944 152

(STM14_2352), t1774 (STM14_1312) and t2782 (STM14_3479) – were upregulated in S. 153

Typhi but downregulated in S. Typhimurium. Given their regulation pattern, these genes may 154

encode uncharacterised virulence factors or be involved in motility in Salmonella. 155

We also analysed the expression profile of S. Typhi specific genes. S. Typhi Ty2 carries 453 156

unique genes relative to S. Typhimurium (representing Ty2 homologues of the 601 S. Typhi 157

specific genes identified in CT18 (36), in addition to 29 Ty2 specific genes (37)). Only two of 158

these genes were significantly regulated by bile exposure in both S. Typhi Ty2 and 129-0238. 159

Both genes, which are upregulated in bile, encode hypothetical proteins: t0349 (STY2749) 160

encodes a GIY-YIG domain containing protein, and t1865 (STY1076) encodes a homologue 161

of the NleG family of T3SS effectors (38, 39). Neither S. Typhi isolate demonstrated altered 162

expression of genes encoding the Vi antigen or of the typhoid toxin in bile. 163

Bile influences SPI-1 expression and Salmonella invasion 164

The most marked differences between S. Typhi and S. Typhimurium in response to bile was 165

in the expression of SPI-1-associated genes. The majority of genes within the SPI-1 166

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

9

pathogenicity island, in addition to the SPI-1 regulators rtsA and rtsB, and effector genes 167

carried outside SPI-1 (sopD), were significantly upregulated in S. Typhi Ty2 and 129-0238 168

but significantly downregulated in S. Typhimurium (Table 3; Figure 3A). Noticeably, S. 169

Typhi 129-0238 exhibited significantly elevated expression of SPI-1 genes relative to S. 170

Typhi Ty2 (Table 3; Figure 3A). 171

To determine if changes in SPI-1 gene expression correlated with changes at the protein 172

level, we compared the intracellular levels of the SPI-1 translocon proteins SipC, SipD, and 173

the SPI-1 effectors SopE (for S. Typhi) or SopB (for S. Typhi and S. Typhimurium) from 174

each strain grown in the absence or presence of bile. Additional S. Typhi strains were also 175

included to further expand and validate these findings, namely the RpoS+ S. Typhi reference 176

strain CT18 (37), and an additional H58 isolate, ERL12148 which belongs to a different 177

sublineage of H58 than 129-0238 (21). All S. Typhi strains tested (Ty2, CT18, 129-0238, 178

ERL12148) showed increased levels of SPI-1 proteins, with the H58 strains demonstrating 179

the largest increases in SPI-1 protein expression in bile (Figure 3B, Figure S2). Conversely S. 180

Typhimurium 14028 showed decreased levels of SopB, SipD and SipC following growth in 181

bile (Figure 3B, Figure S2); as S. Typhimurium 14028 lacks SopE, its lanes (Tm) in the SopE 182

panel are not shown. 183

Given the significant effect of bile on SPI-1 expression, we investigated the impact of bile on 184

epithelial cell invasion. In line with previous findings (14), S. Typhimurium exposed to bile 185

demonstrated significantly reduced invasion, achieving an invasion rate approximately 90% 186

lower than S. Typhimurium grown in the absence of bile (Figure 3C). In contrast, all S. Typhi 187

strains tested demonstrated significantly increased invasion following bile exposure, with 188

Ty2 and CT18 displaying an approximate 2-fold increase in the number of intracellular 189

bacteria at 2 h post-infection, and both H58 isolates demonstrating even higher increases in 190

invasion (between 4-16 fold) (Figure 3C, Figure S2). A SPI-1 deficient strain of S. Typhi Ty2 191

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

10

(ΔinvA) did not invade HeLa cells in the presence of bile, indicating that the increased 192

invasiveness of S. Typhi in bile is SPI-1 dependent (Figure S2). 193

Transcriptional regulation of SPI-1 regulators in bile 194

Given the striking difference in SPI-1 expression between S. Typhi and S. Typhimurium in 195

response to bile, we determined where and how SPI-1 regulation differs between the two 196

serovars. The central regulators governing SPI-1 expression are HilA, often termed the 197

master SPI-1 regulator, and HilD, which is the dominant regulator of HilA (3, 40). The RNA-198

Seq and RT-qPCR data show that the mRNA levels of these regulators significantly decrease 199

in S. Typhimurium in response to bile, but significantly increase in response to bile in the S. 200

Typhi strains (Table 2). 201

In order to determine if these changes are mediated by transcriptional regulation of these 202

genes, we constructed hilA and hilD lacZ chromosomal transcriptional reporters in S. 203

Typhimurium 14028 and S. Typhi Ty2 (41). The reporter activity was determined by β-204

galactosidase assay following growth to late exponential phase in LB with or without 3% 205

bile. In S. Typhimurium expression of hilA is significantly reduced in the presence of bile, 206

with expression almost 20 fold lower, while expression of hilD is unchanged (Figure 4). In 207

contrast, expression of hilA in S. Typhi significantly increases in bile, with expression over 3 208

times higher, whilst hilD expression is only modestly increased (Figure 4). Taken together, 209

these results indicate that hilA is transcriptionally regulated by bile in both S. Typhi and S. 210

Typhimurium, whilst hilD is not subject to transcriptional regulation. 211

The seeming absence of hilD transcriptional regulation in bile (Figure 4) is at odds with the 212

significant changes in mRNA levels observed (Table 2). One explanation is that hilD:lacZ 213

reporter strains do not account for HilD-mediated autoregulation, as the chromosomal 214

reporter strains were made in a hilD background. HilD autoregulation has previously been 215

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

11

reported in S. Typhimurium (42), but has not been characterised in S. Typhi. To determine if 216

HilD autoregulation could account for transcriptional changes of hilD in bile in S. Typhi, the 217

hilD:lacZ S. Typhi Ty2 reporter strain was transformed with a plasmid expressing HilD or an 218

empty vector control, and reporter activity assessed by β-galactosidase assay following 219

growth in LB. hilD expression from the strain complemented with HilD was significantly 220

higher than hilD expression from both the reporter strain alone and the reporter carrying the 221

empty vector (Figure 5), indicating that in S. Typhi HilD positively regulates its own 222

transcription, either directly or indirectly. 223

Bile influences HilD stability 224

Given that expression of hilA, a gene directly regulated by HilD, significantly increases in 225

bile, we investigated if HilD is post-transcriptionally regulated by bile in S. Typhi. Previous 226

studies have shown that in S. Typhimurium, HilD stability is markedly decreased in the 227

presence of bile, with a reported half-life almost 4 times shorter in LB supplemented with 3% 228

bile, than in LB alone (23). To determine the effect of bile on HilD stability in S. Typhi, S. 229

Typhi Ty2 was transformed with constitutively expressed HA-tagged HilD (from S. Typhi 230

Ty2), subcultured in the presence or absence of bile, and samples taken at regular intervals 231

following the inhibition of protein synthesis. Importantly the HA-tagged HilD used in these 232

studies was functional (Figure 5), indicating that the HA tag used does not disrupt HilD 233

structure or activity. In LB the half-life of HilD was 14 min, while in bile the half-life of HilD 234

increased to 40 min, indicating that HilD is approximately three times more stable in the 235

presence of bile in S. Typhi (Figure 6A). 236

HilD is highly conserved between S. Typhi and S. Typhimurium (>99% identity; 2 amino 237

acid changes). Since HilD has previously been shown to be less stable in bile in S. 238

Typhimurium (23), we next determined if this difference in stability was due to intrinsic 239

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

12

differences between HilD between the serovars, or rather due to differences in factors that act 240

on HilD and influence its stability. To investigate this, we determined the stability of HA-241

tagged HilD from S. Typhimurium 14028 expressed in S. Typhi Ty2. As for S. Typhi HilD, S. 242

Typhimurium HilD was three times more stable in bile, with a recorded half-life increasing 243

from 8 min in LB, to 21 min (Figure 6B). 244

Although several factors have been reported to post-transcriptionally regulate HilD (e.g. 245

HilE, CsrA, GreE/GreB, FliZ, Hfq, RNase E (3, 43, 44)), only two have been described to 246

directly influence HilD protein stability: the protease Lon, which degrades HilD (45), and the 247

acetyltransferase Pat, which acetylates HilD to increase stability whilst decreasing DNA 248

binding (46). To determine if these factors were involved in mediating HilD stability in bile 249

in S. Typhi Ty2, deletions were constructed and HilD stability determined as previously. 250

Unfortunately, a Δlon Ty2 strain had severe growth defects and could not be tested. Although 251

HilD stability was decreased in a Δpat Ty2 strain, in line with previous findings in S. 252

Typhimurium (46, 47), stability of HilD was still increased in the presence of bile, increasing 253

from 4 min in LB to 13 min in the presence of bile (Figure S3), indicating that Pat-mediated 254

acetylation of HilD is not responsible for the increased stability in bile. Overall, our data 255

suggest that factors responsible for governing the stability of HilD in response to bile (other 256

than Pat) differ between S. Typhi and S. Typhimurium. 257

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

13

DISCUSSION 258

Transcriptomic analysis of S. Typhimurium and S. Typhi strains grown in LB or 3% bile 259

permitted the identification of similarities and differences in each serovars’ response to bile. 260

Significant differences were observed in the regulation of the invasion-associated SPI-1 T3SS 261

and in motility genes between non-typhoidal and typhoidal serovars. S. Typhi strains 262

significantly upregulated these processes, and displayed a significant increase in T3SS-263

dependent invasion in bile, a response akin to other enteric pathogens (13), including Vibrio 264

parahaemolyticus (48), Vibrio cholera (49, 50), and Shigella (51, 52). All S. Typhi strains 265

tested (Ty2, CT18 and two H58 clinical isolates) demonstrated significantly increased 266

invasion in bile, strongly suggesting that this is a common response of S. Typhi to bile. 267

It is interesting to consider why S. Typhi and S. Typhimurium have such disparate responses 268

to bile. During infection, Salmonella encounters bile within the small intestine, and in the 269

case of S. Typhi, within the gallbladder. Following the observation that S. Typhimurium 270

invasion was significantly repressed in the presence of bile (14), a model was proposed that 271

S. Typhimurium uses bile concentration as a means to sense proximity to the intestinal 272

epithelium; in the lumen where bile concentration is highest, SPI-1 expression would be 273

repressed, as the bacteria get closer to the intestinal cells, bile concentration would decrease, 274

leading to SPI-1 expression and invasion (14). Within the context of this model however, S. 275

Typhi would be less invasive when in close contact with the intestinal epithelium, which is 276

consistent with the limited intestinal inflammatory responses induced by S. Typhi (1). 277

Moreover, S. Typhi has a unique site of infection – the gallbladder (7, 9). One of the 278

mechanisms by which S. Typhi has been proposed to persist within the gallbladder is via 279

direct invasion of gallbladder epithelial cells (53, 54); bile-induced increases in SPI-1 280

expression and invasiveness may therefore promote S. Typhi invasion and colonisation of the 281

gallbladder epithelium. Alternatively, as S. Typhi carriage is closely associated with the 282

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

14

presence of gallstones, it is believed that S. Typhi forms biofilms on gallstone surfaces (7, 283

55). Biofilm formation on gallstones depends on several factors including the presence of 284

flagellar filaments (56), increased flagellar expression may therefore also promote biofilm 285

formation. As such, increases in expression of SPI-1 and motility associated genes in bile 286

may promote S. Typhi colonisation of the gallbladder, and therefore reflect adaptation to this 287

environment. 288

In terms of understanding how S. Typhi and S. Typhimurium differ with regards to SPI-1 289

expression in bile, our results, in combination with previous findings (23), demonstrate that 290

HilD is differentially regulated by bile at the level of protein stability (consistent with the 291

idea that HilD is largely controlled at the post-transcriptional level (40)), resulting in 292

significant differences in the expression of downstream genes, including the SPI-1 master 293

regulator, hilA (Figure 7). The factor(s) responsible for mediating changes in HilD stability in 294

response to bile remains to be established, however this response does not appear to rely on 295

Lon (23) or Pat (this study). A recent transposon screen which aimed to identify factors 296

responsible for bile-mediated SPI-1 repression in S. Typhimurium failed to identify any 297

regulatory factor other than HilD (23). There are several reasons why such an approach may 298

have failed, including the involvement of essential genes or redundancy. Unfortunately 299

attempts to further identify regulatory mechanisms in S. Typhi are confounded by the limited 300

characterisation of SPI-1 regulatory processes within S. Typhi. The overall effect of bile on 301

invasion between S. Typhi and S. Typhimurium may also not be entirely regulatory; for 302

example the translocon protein SipD has been reported to interact with bile salts (57), but 303

SipD is one of several T3SS-associated proteins reported to be 'differentially evolved’ (as 304

determined by non-synonymous amino acid changes) between typhoidal and non-typhoidal 305

serovars, which results in functional differences (58). Importantly, in Shigella flexneri, 306

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

15

interaction of deoxycholate or other bile salts with the SipD homologue, IpaD, promotes the 307

recruitment of the translocator protein, IpaB, ‘readying’ the T3SS for secretion (59, 60). 308

Our results also demonstrate that strains belonging to the H58 S. Typhi lineage (129-0238 309

and ERL12148) display significantly increased responses to bile when compared to S. Typhi 310

reference strains (Ty2 and CT18). When considering chronic carriage such responses may be 311

advantageous, by increasing the potential of H58 strains to colonise the gallbladder, 312

increasing bacterial burden and subsequently increasing transmission. However, it is 313

currently unknown if this reflects differences between recently isolated clinical strains when 314

compared to more laboratory-adapted reference strains, or is instead due to intrinsic 315

difference in H58 strains compared to other S. Typhi haplotypes. H58 isolates have 44 non-316

synonymous single nucleotide polymorphisms (SNPs) which are not found within the S. 317

Typhi reference strain CT18 (21), including several SNPs within the Csr system (sirA 318

(L63F), csrB (155G>A), csrD (A620V)), which is a known regulator of SPI-1 (61). 319

Interestingly, significant phenotypic differences in bile were also observed between the two 320

H58 strains investigated. Further comparisons of H58 strains would be required to determine 321

if the phenotypic differences observed are sublineage-specific or simply reflect diversity 322

within the H58 group. 323

In conclusion, our results confirm that bile is a key regulator of gene expression in 324

Salmonella, influencing the expression of almost 10% of the genome, including genes 325

associated with virulence, motility and metabolism. These findings add to the characterisation 326

of S. Typhi responses to bile (30, 62), which may ultimately help explain the mechanisms by 327

which S. Typhi induces chronic carriage (13). 328

329

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

16

MATERIALS AND METHODS 330

Bacterial strains, growth conditions and plasmid construction 331

The strains and plasmids used in this study are listed in Table 4. Salmonella were routinely 332

grown in LB Lennox (Sigma-Aldrich) at 37°C / 200 rpm. Ox bile (3% w/v) (Sigma-333

Aldrich/Merck-Millipore) was supplemented as indicated. 334

All oligonucleotides used in this study are listed in Table S1. The ΔinvA and Δpat S. Typhi 335

Ty2 deletion strains were constructed via lambda red, as previously described (63, 64). 336

Strains with chromosomal integration of the lacZ gene were also constructed via lambda red 337

recombination as described (41). Correct integration of introduced cassettes was validated by 338

PCR. 339

To create HA tagged HilD, pWSK29-Spec-4HA (64) was amplified with a reverse primer 340

containing a PacI digestion site, and HilD was amplified from both S. Typhimurium and S. 341

Typhi with primers containing NotI and PacI restriction sites. Both products were digested, 342

and HilD cloned into the existing NotI site and the introduced PacI site of pWSK29-Spec-343

4HA, resulting in constitutively expressed C-terminally tagged HilD-4HA. Plasmid 344

construction was validated by sequencing. 345

Cell culture and HeLa invasion assays 346

HeLa cells (ATCC) were maintained in Dulbecco’s Modified Eagle medium supplemented 347

with 10% foetal bovine serum (FBS) (Sigma-Aldrich) in a 5% CO2 at 37°C. The cells were 348

authenticated via short tandem repeat profiling in February 2016 (Microsynth). 349

Invasiveness of strains was determined by gentamicin protection assays, as previously 350

described (64). Briefly, Salmonella strains were cultured overnight at 37°C / 200 rpm in LB 351

or LB supplemented with 3% bile before subculturing 1:33 in LB or LB 3% bile until late 352

exponential phase (OD600 ~1.8), when SPI-1 expression is induced (18) (data not shown). To 353

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

17

prevent bile-mediated cell lysis, bacteria were washed twice in LB before addition to cells at 354

an MOI 100:1. As S. Typhi is less invasive than S. Typhimurium (65), S. Typhi infections 355

were performed for 1 h, and S. Typhimurium for 15 min, prior to the addition of gentamicin, 356

unless otherwise indicated. At indicated time points, cells were lysed, serially diluted, and 357

plated to enumerate intracellular CFU. 358

RNA extraction 359

Salmonella were cultured overnight in LB or LB supplemented with 3% bile (w/v) before 360

subculturing 1:33 until late exponential phase (OD600 ~1.8). 6 x 108 bacteria were incubated 361

in RNAprotect (Qiagen) at room temperature (RT) for 5 min. Bacteria were digested with 362

lysozyme (15 mg / ml) and proteinase K for 20 min at RT, and RNA extracted using the 363

RNeasy Mini Kit (Qiagen) as per manufacturer’s instructions. RNA extractions for RNA-Seq 364

were performed in duplicate then pooled, over three biological repeats. RNA extractions for 365

quantitative reverse transcription PCR (RT-qPCR) were performed in triplicate over three 366

biological repeats. RNA samples for RNA-Seq and RT-qPCR were extracted independently 367

of each other. 368

RNA sequencing and data analysis 369

For RNA sequencing, mRNA libraries were multiplexed and prepared by utilisation of the 370

Illumina TruSeq protocol followed by sequencing via paired-end methodology on the 371

Illumina HiSeq version 4 platform. Each lane of Illumina sequence was assessed for quality 372

on the basis of adapter contamination, average base read quality and any unusual G-C bias 373

using FastQC. The median Phred score for all samples was >34. To permit comparison 374

between strains, sequenced reads for each strain were mapped to the Ty2 genome 375

(NC_004631) using the Rockhopper tool (66) with default parameters (Data S1-3). The read 376

alignment coverage for each sample can be found in Table S2. The threshold for 377

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

18

differentially expressed genes was gated as those displaying >2 fold change in expression in 378

3% bile compared to LB alone, and with an adjusted p value (q value) < 0.05. 379

GO term enrichment for differentially regulated genes was performed with Panther (67) using 380

the S. Typhimurium GO annotation, whilst KEGG pathway analysis was performed with the 381

GAGE R package (68) (R 3.3.1), using the S. Typhi (stt) KEGG annotation. The 382

VennDiagram (69) and gplots R packages were used for data visualisation. 383

Quantitative reverse transcription PCR (RT-qPCR) 384

2 µg of RNA was treated with DNase (Promega) prior to reverse transcription with M-MLV 385

reverse transcriptase (Promega) according to manufacturer’s recommendations. Fast SYBR 386

Green Master Mix (Applied Biosystems) was used for qPCR reactions alongside the Applied 387

Biosystems StepOnePlus system. 20 ng of cDNA was used per reaction, and forward and 388

reverse primers (Table S1) used at final concentration of 0.2 µM. Samples without reverse 389

transcription were included as negative controls. The housekeeping gene, ftsZ, was used as 390

the reference gene as it was determined to be least variable gene between strains and between 391

LB with and without 3% bile. qPCR reactions were performed in duplicate on triplicate 392

samples over three biological replicates. 393

SPI-1 protein expression and stability assays 394

To determine expression of SPI-1 proteins, Salmonella were subcultured in the absence or 395

presence of 3% ox-bile to late exponential phase. 1 mL of culture was pelleted and re-396

suspended in 2X SDS loading buffer (1M Tris pH 6.8, 2% SDS, 20% glycerol, 5% β-397

mercaptoethanol, bromophenol blue) in proportion to OD600. To determine HilD stability, 398

Salmonella strains previously transformed with 4HA-tagged constructs were subcultured in 399

10 ml LB with or without the addition of 3% ox-bile until late exponential phase. The OD600 400

was recorded, and chloramphenicol (30 µg/ml) added to inhibit protein synthesis. 1 ml 401

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

19

bacteria were pelleted and re-suspended in 2X SDS loading buffer in proportion to OD600. 402

The cultures were incubated at 37°C / 200 rpm, and 1 ml samples were taken at required time 403

points. Samples were heated at 95°C for 10 min. Whole cell samples were subject to Western 404

blotting, using an anti-HA antibody to detect the protein of interest, and DnaK as a loading 405

control. Following imaging, band density was quantified using ImageJ, and half-life (in 406

minutes) calculated using the equation: (t x ln(2)) / (ln(No/Nf)), where t equals time elapsed 407

between measurements (in minutes), N0 equals the initial amount, and Nf equals the final 408

amount (23). To determine changes in SPI-1 proteins in bile, band density was quantified 409

using ImageJ, levels of SPI-1 proteins were normalised to the corresponding DnaK value, and 410

fold change in bile relative to LB calculated. 411

SDS-PAGE and Western blotting 412

Proteins were separated on 12% acrylamide gels followed by semi-dry transfer on to PVDF 413

membrane (GE Healthcare). Membranes were blocked in 5% milk in PBS + 0.05% Tween-20 414

(Sigma-Aldrich), and probed with either anti-DnaK 8E2/2 (1:10000) (Enzo Life Sciences 415

#ADI-SPA-880), anti-HA HA-7 (1:1000) (Sigma #H3663), anti-SipC, anti-SipD, anti-SopB, 416

or anti-SopE (1:5000) (V. Koronakis, University of Cambridge) primary antibodies, followed 417

by HRP-conjugated secondary antibody (1:10000) (Jackson ImmunoResearch). 418

Chemiluminescence following the addition of EZ-ECL reagent (Geneflow) was detected 419

using the LAS-3000 imager (Fuji). 420

β-galactosidase assays 421

β-galactosidase assays were performed as previously described (70). Salmonella strains were 422

grown in SPI-1 inducing conditions with or without the addition of 3% ox bile. The OD600 423

was recorded, and 1 ml of culture pelleted and resuspend in 1 ml Z buffer (0.06M Na2HPO4, 424

0.04M NaH2PO4, 0.01M KCl, 0.001M MgSO4 and 0.05M β-mercaptoethanol, pH 7). WT 425

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

20

strains were used as negative controls. Samples were permeabilised with the addition of 0.1% 426

SDS and chloroform, and vortexed for 2 min. 20 µl of prepared sample was added to 180 µl 427

Z buffer in a 96 well microplate, and 2-Nitrophenyl β-D-galactopyranoside (ONPG) substrate 428

(4 mg/ml in Z buffer) added. Plates were incubated at RT, then the reaction stopped with the 429

addition of 1M Na2CO3. The absorbance of the samples was measured at 405 nm and 540 nm 430

using a FLUOStar Omega plate reader (BMG Labtech). 431

Statistical analysis 432

Statistical tests were performed using GraphPad Prism (Version 7.00) for Windows 433

(GraphPad Software, San Diego, California, USA). All data are expressed as mean ± SD. 434

Significance (p < 0.05) was determined by unpaired t-test or ANOVA, with correction for 435

multiple comparisons when required. 436

437

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

21

ACKNOWLEDGEMENTS 438

We are grateful to Gordon Dougan (Sanger Institute) for providing the S. Typhi strains used 439

in this study, to Michael Hensel for providing the p3138 template plasmid for construction of 440

reporter strains via lambda red, and to Vassilis Koronakis (University of Cambridge) for 441

providing the anti-SipC, anti-SipD, anti-SopB and anti-SopE antibodies. RJ is supported by 442

an MRC Centre for Molecular Bacteriology and Infection Grant, ref: MR/J006874/1. GF is 443

supported by a Wellcome Trust Investigator grant. 444

445

446

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

22

REFERENCES 447

1. Dougan G, Baker S. 2014. Salmonella enterica Serovar Typhi and the Pathogenesis of 448

Typhoid Fever. Annu Rev Microbiol 68:317–36. 449

2. McGhie EJ, Brawn LC, Hume PJ, Humphreys D, Koronakis V. 2009. Salmonella 450

takes control: effector-driven manipulation of the host. Curr Opin Microbiol 12:117–451

24. 452

3. Fàbrega A, Vila J. 2013. Salmonella enterica serovar Typhimurium skills to succeed 453

in the host: virulence and regulation. Clin Microbiol Rev 26:308–41. 454

4. Altier C. 2005. Genetic and Environmental Control of Salmonella Invasion. J 455

Microbiol 43:85–92. 456

5. Parry C, Dougan G. 2002. Typhoid Fever. N Engl J Med 347:1770–1782. 457

6. Crump JA, Luby SP, Mintz ED. 2004. The global burden of typhoid fever. Bull 458

World Health Organ 82:346–53. 459

7. Gunn JS, Marshall JM, Baker S, Dongol S, Charles RC, Ryan ET. 2014. 460

Salmonella chronic carriage: epidemiology, diagnosis, and gallbladder persistence. 461

Trends Microbiol 22:648–655. 462

8. Gal-Mor O, Boyle EC, Grassl GA. 2014. Same species, different diseases: how and 463

why typhoidal and non-typhoidal Salmonella enterica serovars differ. Front Microbiol 464

5:391. 465

9. Gonzalez-Escobedo G, Marshall JM, Gunn JS. 2011. Chronic and acute infection of 466

the gall bladder by Salmonella Typhi: understanding the carrier state. Nat Rev 467

Microbiol 9:9–14. 468

10. Begley M, Gahan C, Hill C. 2005. The interaction between bacteria and bile. FEMS 469

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

23

Microbiol Rev 29:625–651. 470

11. Gunn JS. 2000. Mechanisms of bacterial resistance and response to bile. Microbes 471

Infect 2:907–913. 472

12. van Velkinburgh JC, Gunn JS. 1999. PhoP-PhoQ-regulated loci are required for 473

enhanced bile resistance in Salmonella spp. Infect Immun 67:1614–22. 474

13. Sistrunk JR, Nickerson KP, Chanin RB, Rasko DA, Faherty CS. 2016. Survival of 475

the Fittest: How Bacterial Pathogens Utilize Bile To Enhance Infection. Clin Microbiol 476

Rev 29:819–836. 477

14. Prouty AM, Gunn JS. 2000. Salmonella enterica Serovar Typhimurium Invasion Is 478

Repressed in the Presence of Bile. Infect Immun 68:6763–6769. 479

15. Prouty AM, Brodsky IE, Manos J, Belas R, Falkow S, Gunn JS. 2004. 480

Transcriptional regulation of Salmonella enterica serovar Typhimurium genes by bile. 481

FEMS Immunol Med Microbiol 41. 482

16. Antunes LCM, Wang M, Andersen SK, Ferreira RBR, Kappelhoff R, Han J, 483

Borchers CH, Finlay BB. 2012. Repression of Salmonella enterica phoP expression 484

by small molecules from physiological bile. J Bacteriol 194:2286–96. 485

17. Hernández SB, Cota I, Ducret A, Aussel L, Casadesús J. 2012. Adaptation and 486

Preadaptation of Salmonella enterica to Bile. PLoS Genet 8:e1002459. 487

18. Kröger C, Colgan A, Srikumar S, Händler K, Sivasankaran SK, Hammarlöf DL, 488

Canals R, Grissom JE, Conway T, Hokamp K, Hinton JCD. 2013. An infection-489

relevant transcriptomic compendium for Salmonella enterica Serovar Typhimurium. 490

Cell Host Microbe 14:683–95. 491

19. Sengupta C, Ray S, Chowdhury R. 2014. Fine tuning of virulence regulatory 492

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

24

pathways in enteric bacteria in response to varying bile and oxygen concentrations in 493

the gastrointestinal tract. Gut Pathog 6:38. 494

20. Sabbagh SC, Forest CG, Lepage C, Leclerc J-M, Daigle F. 2010. So similar, yet so 495

different: uncovering distinctive features in the genomes of Salmonella enterica 496

serovars Typhimurium and Typhi. FEMS Microbiol Lett 305:1–13. 497

21. Wong V, Baker S, Pickard D, Parkhill J, Page A, Feasey N, Kingsley R, Thomson 498

N, Keane J, Weill F-X, Edwards D, Hawkey J, Harris SR, Mather AE, Cain AK, 499

Hadfield J, Hart PJ, Thieu NTV, Klemm EJ, Glinos DA, Breiman RF, Watson 500

CH, Kariuki S, Gordon MA, Heyderman RS, Okoro C, Jacobs J, Lunguya O, 501

Edmunds WJ, Msefula C, Chabalgoity JA, Kama M, Jenkins K, Dutta S, Marks 502

F, Campos J, Thompson C, Obaro S, MacLennan CA, Dolecek C, Keddy KH, 503

Smith AM, Parry CM, Karkey A, Mulholland EK, Campbell JI, Dongol S, 504

Basnyat B, Dufour M, Bandaranayake D, Naseri TT, Singh SP, Hatta M, Newton 505

P, Onsare RS, Isaia L, Dance D, Davong V, Thwaites G, Wijedoru L, Crump JA, 506

De Pinna E, Nair S, Nilles EJ, Thanh DP, Turner P, Soeng S, Valcanis M, 507

Powling J, Dimovski K, Hogg G, Farrar J, Holt KE, Dougan G. 2015. 508

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of 509

Salmonella Typhi identifies inter- and intracontinental transmission events. Nat Genet 510

47:632–639. 511

22. Wong V, Baker S, Connor T, Pickard D, Page A, Dave J, Murphy N, Holliman R, 512

Sefton A, Millar M, Dyson ZA, Dougan G, Holt K, International Typhoid 513

Consortium IT. 2016. An extended genotyping framework for Salmonella enterica 514

serovar Typhi, the cause of human typhoid. Nat Commun 7:12827. 515

23. Eade CR, Hung C-C, Bullard B, Gonzalez-Escobedo G, Gunn JS, Altier C. 2016. 516

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

25

Bile Acids Function Synergistically to Repress Invasion Gene Expression in 517

Salmonella by Destabilizing the Invasion Regulator HilD. Infect Immun 84:2198–518

2208. 519

24. Bishop RE. 2005. The lipid A palmitoyltransferase PagP: molecular mechanisms and 520

role in bacterial pathogenesis. Mol Microbiol 57:900–912. 521

25. Perkins TT, Davies MR, Klemm EJ, Rowley G, Wileman T, James K, Keane T, 522

Maskell D, Hinton JCD, Dougan G, Kingsley RA. 2013. ChIP-seq and 523

transcriptome analysis of the OmpR regulon of Salmonella enterica serovars Typhi 524

and Typhimurium reveals accessory genes implicated in host colonization. Mol 525

Microbiol 87:526–538. 526

26. Lawhon SD, Maurer R, Suyemoto M, Altier C. 2002. Intestinal short-chain fatty 527

acids alter Salmonella typhimurium invasion gene expression and virulence through 528

BarA/SirA. Mol Microbiol 46:1451–1464. 529

27. Antunes LCM, Andersen SK, Menendez A, Arena ET, Han J, Ferreira RBR, 530

Borchers CH, Finlay BB. 2011. Metabolomics reveals phospholipids as important 531

nutrient sources during Salmonella growth in bile in vitro and in vivo. J Bacteriol 532

193:4719–25. 533

28. Denkel LA, Horst SA, Rouf SF, Kitowski V, Böhm OM, Rhen M, Jäger T, Bange 534

F-C. 2011. Methionine Sulfoxide Reductases Are Essential for Virulence of 535

Salmonella Typhimurium. PLoS One 6:e26974. 536

29. Ibanez-Ruiz M, Robbe-Saule V, Hermant D, Labrude S, Norel F. 2000. 537

Identification of RpoS (sigma(S))-regulated genes in Salmonella enterica serovar 538

typhimurium. J Bacteriol 182:5749–56. 539

30. Walawalkar YD, Vaidya Y, Nayak V. 2016. Response of Salmonella Typhi to bile 540

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

26

generated oxidative stress: implication of quorum sensing and persister cell 541

populations. Pathog Dis. 542

31. Robbe-Saule V, Coynault C, Norel F. 1995. The live oral typhoid vaccine Ty21a is a 543

rpoS mutant and is susceptible to various environmental stresses. FEMS Microbiol 544

Lett 126:171–176. 545

32. Kawasaki K, Teramoto M, Tatsui R, Amamoto S. 2012. Lipid A 3′-O-deacylation 546

by Salmonella outer membrane enzyme LpxR modulates the ability of lipid A to 547

stimulate Toll-like receptor 4Biochemical and Biophysical Research Communications. 548

33. Kawano M, Manabe T, Kawasaki K. 2010. Salmonella enterica serovar 549

Typhimurium lipopolysaccharide deacylation enhances its intracellular growth within 550

macrophages. FEBS Lett 584:207–212. 551

34. García-Calderón CB, Casadesús J, Ramos-Morales F. 2007. Rcs and PhoPQ 552

regulatory overlap in the control of Salmonella enterica virulence. J Bacteriol 553

189:6635–44. 554

35. Lei L, Wang W, Xia C, Liu F. 2016. Salmonella Virulence Factor SsrAB Regulated 555

Factor Modulates Inflammatory Responses by Enhancing the Activation of NF-κB 556

Signaling Pathway. J Immunol 196:792–802. 557

36. Parkhill J, Dougan G, James KD, Thomson NR, Pickard D, Wain J, Churcher C, 558

Mungall KL, Bentley SD, Holden MT, Sebaihia M, Baker S, Basham D, Brooks 559

K, Chillingworth T, Connerton P, Cronin A, Davis P, Davies RM, Dowd L, White 560

N, Farrar J, Feltwell T, Hamlin N, Haque A, Hien TT, Holroyd S, Jagels K, 561

Krogh A, Larsen TS, Leather S, Moule S, O’Gaora P, Parry C, Quail M, 562

Rutherford K, Simmonds M, Skelton J, Stevens K, Whitehead S, Barrell BG. 563

2001. Complete genome sequence of a multiple drug resistant Salmonella enterica 564

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

27

serovar Typhi CT18. Nature 413:848–52. 565

37. Deng W, Liou S-R, Plunkett G, Mayhew GF, Rose DJ, Burland V, Kodoyianni V, 566

Schwartz DC, Blattner FR. 2003. Comparative genomics of Salmonella enterica 567

serovar Typhi strains Ty2 and CT18. J Bacteriol 185:2330–7. 568

38. Tobe T, Beatson SA, Taniguchi H, Abe H, Bailey CM, Fivian A, Younis R, 569

Matthews S, Marches O, Frankel G, Hayashi T, Pallen MJ. 2006. An extensive 570

repertoire of type III secretion effectors in Escherichia coli O157 and the role of 571

lambdoid phages in their dissemination. Proc Natl Acad Sci 103:14941–14946. 572

39. Hannemann S, Galán JE, Beek M van den, Blankenberg D, Bouvier D, Čech M. 573

2017. Salmonella enterica serovar-specific transcriptional reprogramming of infected 574

cells. PLOS Pathog 13:e1006532. 575

40. Ellermeier JR, Slauch JM. 2007. Adaptation to the host environment: regulation of 576

the SPI1 type III secretion system in Salmonella enterica serovar Typhimurium. Curr 577

Opin Microbiol 10:24–9. 578

41. Gerlach RG, Hölzer SU, Jäckel D, Hensel M. 2007. Rapid engineering of bacterial 579

reporter gene fusions by using Red recombination. Appl Environ Microbiol 73:4234–580

42. 581

42. Ellermeier CD, Ellermeier JR, Slauch JM. 2005. HilD, HilC and RtsA constitute a 582

feed forward loop that controls expression of the SPI1 type three secretion system 583

regulator hilA in Salmonella enterica serovar Typhimurium. Mol Microbiol 57:691–584

705. 585

43. Gaviria-Cantin T, El Mouali Y, Le Guyon S, Römling U, Balsalobre C, Rüssmann 586

H. 2017. Gre factors-mediated control of hilD transcription is essential for the invasion 587

of epithelial cells by Salmonella enterica serovar Typhimurium. PLOS Pathog 588

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

28

13:e1006312. 589

44. López-Garrido J, Puerta-Fernández E, Casadesús J. 2014. A eukaryotic-like 3’ 590

untranslated region in Salmonella enterica hilD mRNA. Nucleic Acids Res 42:5894–591

906. 592

45. Takaya A, Kubota Y, Isogai E, Yamamoto T. 2004. Degradation of the HilC and 593

HilD regulator proteins by ATP-dependent Lon protease leads to downregulation of 594

Salmonella pathogenicity island 1 gene expression. Mol Microbiol 55:839–852. 595

46. Sang Y, Ren J, Qin R, Liu S, Cui Z, Cheng S, Liu X, Lu J, Tao J, Yao Y-F. 2017. 596

Acetylation regulates protein stability and DNA-binding ability of HilD to modulate 597

Salmonella Typhimurium virulence. J Infect Dis. 598

47. Sang Y, Ren J, Ni J, Tao J, Lu J, Yao Y-F. 2016. Protein Acetylation Is Involved in 599

Salmonella enterica Serovar Typhimurium Virulence. J Infect Dis 213:1836–1845. 600

48. Li P, Rivera-Cancel G, Kinch LN, Salomon D, Tomchick DR, Grishin N V, Orth 601

K, Fenical W, Zhu J, Ochi S, Sasahara T, Hayashi S, Hirai Y, Sakurai J, 602

Shinagawa H, Hattori M, Iida T. 2016. Bile salt receptor complex activates a 603

pathogenic type III secretion system. Elife 5:70–77. 604

49. Gupta S, Chowdhury R. 1997. Bile affects production of virulence factors and 605

motility of Vibrio cholerae. Infect Immun 65:1131–4. 606

50. Alam A, Tam V, Hamilton E, Dziejman M. 2010. vttRA and vttRB Encode ToxR 607

family proteins that mediate bile-induced expression of type three secretion system 608

genes in a non-O1/non-O139 Vibrio cholerae strain. Infect Immun 78:2554–70. 609

51. Pope LM, Reed KE, Payne SM. 1995. Increased protein secretion and adherence to 610

HeLa cells by Shigella spp. following growth in the presence of bile salts. Infect 611

Immun 63:3642–8. 612

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

29

52. Nickerson KP, Chanin RB, Sistrunk JR, Rasko DA, Fink PJ, Barry EM, Nataro 613

JP, Faherty CS. 2017. Analysis of Shigella flexneri Resistance, Biofilm Formation, 614

and Transcriptional Profile in Response to Bile Salts. Infect Immun 85:e01067-16. 615

53. Gonzalez-Escobedo G, Gunn JS. 2013. Gallbladder epithelium as a niche for chronic 616

Salmonella carriage. Infect Immun 81:2920–30. 617

54. Menendez A, Arena ET, Guttman JA, Thorson L, Vallance BA, Vogl W, Finlay 618

BB. 2009. Salmonella infection of gallbladder epithelial cells drives local 619

inflammation and injury in a model of acute typhoid fever. J Infect Dis 200:1703–13. 620

55. Crawford RW, Rosales-Reyes R, Ramírez-Aguilar M de la L, Chapa-Azuela O, 621

Alpuche-Aranda C, Gunn JS. 2010. Gallstones play a significant role in Salmonella 622

spp. gallbladder colonization and carriage. Proc Natl Acad Sci U S A 107:4353–8. 623

56. Prouty AM, Gunn JS. 2003. Comparative analysis of Salmonella enterica serovar 624

Typhimurium biofilm formation on gallstones and on glass. Infect Immun 71:7154–8. 625

57. Wang Y, Nordhues BA, Zhong D, De Guzman RN. 2010. NMR Characterization of 626

the Interaction of the Salmonella Type III Secretion System Protein SipD and Bile 627

Salts ,. Biochemistry 49:4220–4226. 628

58. Eswarappa SM, Janice J, Nagarajan AG, Balasundaram S V, Karnam G, Dixit 629

NM, Chakravortty D. 2008. Differentially evolved genes of Salmonella pathogenicity 630

islands: insights into the mechanism of host specificity in Salmonella. PLoS One 631

3:e3829. 632

59. Olive AJ, Kenjale R, Espina M, Moore DS, Picking WL, Picking WD. 2007. Bile 633

salts stimulate recruitment of IpaB to the Shigella flexneri surface, where it colocalizes 634

with IpaD at the tip of the type III secretion needle. Infect Immun 75:2626–9. 635

60. Stensrud KF, Adam PR, La Mar CD, Olive AJ, Lushington GH, Sudharsan R, 636

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

30

Shelton NL, Givens RS, Picking WL, Picking WD. 2008. Deoxycholate interacts 637

with IpaD of Shigella flexneri in inducing the recruitment of IpaB to the type III 638

secretion apparatus needle tip. J Biol Chem 283:18646–54. 639

61. Martínez LC, Yakhnin H, Camacho MI, Georgellis D, Babitzke P, Puente JL, 640

Bustamante VH. 2011. Integration of a complex regulatory cascade involving the 641

SirA/BarA and Csr global regulatory systems that controls expression of the 642

Salmonella SPI-1 and SPI-2 virulence regulons through HilD. Mol Microbiol 643

80:1637–56. 644

62. Langridge GC, Phan M-D, Turner DJ, Perkins TT, Parts L, Haase J, Charles I, 645

Maskell DJ, Peters SE, Dougan G, Wain J, Parkhill J, Turner AK. 2009. 646

Simultaneous assay of every Salmonella Typhi gene using one million transposon 647

mutants. Genome Res 19:2308–16. 648

63. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal genes in 649

Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–5. 650

64. Johnson R, Byrne A, Berger CN, Klemm E, Crepin VF, Dougan G, Frankel G. 651

2017. The type III secretion system effector SptP of Salmonella enterica serovar 652

Typhi. J Bacteriol 199:e00647-16. 653

65. Bishop A, House D, Perkins T, Baker S, Kingsley RA, Dougan G. 2008. Interaction 654

of Salmonella enterica serovar Typhi with cultured epithelial cells: roles of surface 655

structures in adhesion and invasion. Microbiology 154:1914–26. 656

66. McClure R, Balasubramanian D, Sun Y, Bobrovskyy M, Sumby P, Genco CA, 657

Vanderpool CK, Tjaden B. 2013. Computational analysis of bacterial RNA-Seq data. 658

Nucleic Acids Res 41:e140. 659

67. Mi H, Huang X, Muruganujan A, Tang H, Mills C, Kang D, Thomas PD. 2017. 660

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

31

PANTHER version 11: expanded annotation data from Gene Ontology and Reactome 661

pathways, and data analysis tool enhancements. Nucleic Acids Res 45:D183–D189. 662

68. Luo W, Friedman MS, Shedden K, Hankenson KD, Woolf PJ. 2009. GAGE: 663

generally applicable gene set enrichment for pathway analysis. BMC Bioinformatics 664

10:161. 665

69. Chen H, Boutros PC. 2011. VennDiagram: a package for the generation of highly-666

customizable Venn and Euler diagrams in R. BMC Bioinformatics 12. 667

70. Miller J. 1972. Experiments in molecular genetics. Cold Sprong Harbor Laboratory, 668

New York. 669

670

671

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

32

FIGURE LEGENDS 672

Figure 1. Comparison of pathways differentially regulated by bile between S. Typhi and 673

S. Typhimurium. Overrepresented Gene Ontology (GO) terms within upregulated and 674

downregulated genes following growth in 3% bile for each strain. 675

676

Figure 2. Gene expression in response to bile differs between Salmonella strains. 677

Comparison of genes upregulated and downregulated in response to bile in S. Typhimurium 678

(Tm), S. Typhi Ty2 (Ty2) and S. Typhi 129-0238 (H58). 679

680

Figure 3. The effect of bile on SPI-1 expression and activity. (A) Heatmap showing log2 681

fold change in gene expression for S. Typhimurium (Tm), S. Typhi Ty2 (Ty2) and S. Typhi 682

129-0238 (H58) across the SPI-1 pathogenicity island and for non-SPI-1 carried effectors. 683

Asterisks (*) indicate genes significantly affected by bile across all three strains. (B) Western 684

blots of SipC, SipD and SopE of S. Typhimurium 14028 (Tm), S. Typhi Ty2 (Ty2), and two 685

H58 clinical isolates (ERL12148 and 129-0238) grown in LB with or without 3% bile; SopE 686

panels are not shown for S. Typhimurium 14028, as this strain lacks SopE. DnaK was used as 687

a loading control. A representative blot of two independent repeats is shown. Numbers below 688

panels indicate fold change in density when compared to LB; all bands were normalised to 689

their respective DnaK control prior to comparison. (C) Strains grown in LB or 3% bile to late 690

exponential phase were added to HeLa cells at an MOI 100 for 30 min. The percentage of 691

intracellular bacteria at 2 h post-infection relative to the inoculum added is shown. n=3, error 692

bars show SD. Invasion rates of strains were compared by t-test (** = P < 0.01, *** = P < 693

0.001). 694

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

33

Figure 4. Effect of bile on hilA and hilD transcription in Salmonella. The reporter activity 695

(β-galactosidase units) of hilA:lacZ and hilD:lacZ in S. Typhimurium 14028 (A & B) and S. 696

Typhi Ty2 (C & D) following growth to late exponential phase in LB in the presence or 697

absence of bile. n=3, error bars show SD. Reporter activity between strains was compared by 698

t-test (* = P < 0.05, *** = P < 0.001). 699

700

Figure 5. HilD autoregulation in S. Typhi. The reporter activity of a S. Typhi Ty2 701

hilD:lacZ chromosomal transcriptional reporter strain complemented with HilD (pWSK29-702

Spec HilD-4HA (HilD)) or an empty vector control (pWSK29-Spec (EV)), was determined 703

by β-galactosidase assay following growth in LB. n=3, error bars show SD. Reporter activity 704

between strains was compared by one way ANOVA (*** = P < 0.001). 705

706

Figure 6. Bile promotes HilD stability in S. Typhi. WT S. Typhi Ty2 constitutively 707

expressing C-terminally 4HA-tagged HilD from (A) S. Typhi Ty2 or (B) S. Typhimurium 708

14028 was grown in LB with or without bile. 30 µg/ml chloramphenicol was added to stop 709

protein synthesis, and samples were collected every 10 min. HilD levels were determined via 710

Western blotting using an anti-HA antibody, and DnaK used as a loading control. A 711

representative blot of three independent repeats is shown. Half-life measurements are 712

averaged from three independent repeats, and standard deviation is shown. 713

714

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

34

Figure 7. Proposed model of how bile influences SPI-1 expression in S. Typhi. (A) HilD 715

is at the top of the SPI-1 regulatory hierarchy, where it regulates its own expression and the 716

expression of HilA. HilD also regulates expression of the additional regulators HilC and 717

RtsA, which also control HilA expression. (B) In the absence of bile the turnover of HilD is 718

high, the expression of hilD is at a basal level and as a result the expression of hilA is low (C) 719

In the presence of bile HilD is more stable, leading to enhanced expression of hilD, hilA and 720

thus SPI-1. 721

722 on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

35

TABLES 723

Table 1. Genes upregulated by bile in all strains 724

Log2 fold

change

Name Locus

tag

Product Tm Ty2 H58

fadI t0475 3-ketoacyl-CoA thiolase 4.12 2.55 2.52

fadJ t0476 multifunctional fatty acid oxidation complex subunit alpha 3.32 2.06 2.17

fadE t2541 acyl-CoA dehydrogenase 7.44 4.70 4.18

fadB t3315 multifunctional fatty acid oxidation complex subunit alpha 7.52 2.92 1.57

fadA t3316 3-ketoacyl-CoA thiolase 7.66 2.88 1.57

actP t4179 acetate permease 3.41 1.58 1.27

- t4180 hypothetical protein 3.44 1.72 1.32

acs t4181 acetyl-CoA synthetase 3.91 2.11 1.31

acnA t1625 aconitate hydratase 3.18 1.81 1.59

argT t0509 lysine-arginine-ornithine-binding periplasmic protein 3.36 2.29 1.33

argD t1182

bifunctional succinylornithine transaminase/acetylornithine

transaminase 5.61 2.72 1.20

- t0677 gentisate 1,2-dioxygenase 2.51 3.91 3.38

- t0678 FAA-hydrolase-family protein 2.09 3.21 2.87

- t0679 glutathione-S-transferase-family protein 2.09 2.89 2.49

- t0680 salicylate hydroxylase 1.27 2.09 2.03

- t1787 oxidoreductase 3.62 3.53 1.32

- t1789 hypothetical protein 3.17 4.04 1.44

- t1790 N-acetylneuraminic acid mutarotase 2.78 4.07 1.32

gabT t2687 4-aminobutyrate aminotransferase 5.18 2.93 1.74

msrA t4462 methionine sulfoxide reductase A 1.68 1.67 1.30

725

726

Table 2. Log2 fold change in gene expression determined by RNA-Seq and RT-qPCR 727

RNA-Seq RT-qPCR

Gene 14028 Ty2 129-0238 14028 Ty2 129-0238

hilD -4.08 1.23 3.15 -3.48

(± 0.71)

1.42

(± 0.25)

2.44

(± 0.73)

hilA -6.98 1.54 3.67 -6.51

(± 0.64)

1.71

(± 0.39)

3.37

(± 0.39)

prgH -6.36 1.57 4.02 -6.00

(± 0.74)

1.68

(± 0.73)

4.00

(± 0.48)

sopB -6.95 1.11 4.21 -3.85

(± 0.44)

1.38

(± 0.59)

4.13

(± 0.27)

flhD -1.72 1.05 1.33 -1.25

(± 0.43)

1.93

(± 0.38)

2.31

(± 1.13)

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

36

flgA -1.29 1.37 1.70 -0.98

(± 0.27)

1.99

(± 0.44)

1.37

(± 0.84)

fadE 7.44 4.70 4.18 3.55

(± 2.13)

3.75

(± 0.16)

4.75

(± 0.09)

acs 3.91 2.11 1.31 2.03

(± 1.77)

0.87

(± 0.67)

2.37

(± 0.59)

± indicates standard deviation 728

729

Table 3. Genes downregulated in S. Typhimurium and upregulated in S. Typhi in bile 730

Log2 fold change

Name Locus tag Product Tm Ty2 H58

fliO t0899 flagellar biosynthesis protein FliO -1.87 1.57 1.35

fliN t0900 flagellar motor switch protein FliN -1.55 1.44 1.62

fliM t0901 flagellar motor switch protein FliM -1.71 1.40 1.71

fliL t0902 flagellar basal body protein FliL -1.74 1.41 1.78

fliK t0903 flagellar hook-length control protein -1.67 1.33 2.08

fliJ t0904 flagellar biosynthesis chaperone -1.37 1.43 2.25

fliI t0905 flagellum-specific ATP synthase -1.43 1.25 1.69

fliH t0906 flagellar assembly protein H -1.45 1.41 1.57

fliG t0907 flagellar motor switch protein G -1.44 1.34 1.53

fliF t0908 flagellar MS-ring protein -1.89 1.32 1.41

fliE t0909 flagellar hook-basal body protein FliE -2.49 1.76 2.01

flhD t0952 transcriptional activator FlhD -1.72 1.05 1.33

flgJ t1738 flagellar rod assembly protein/muramidase FlgJ -1.56 1.30 1.38

flgI t1739 flagellar basal body P-ring biosynthesis protein

FlgA -1.69 1.41 1.39

flgH t1740 flagellar basal body L-ring protein -1.71 1.42 1.68

flgC t1745 flagellar basal body rod protein FlgC -1.86 1.39 1.79

flgB t1746 flagellar basal-body rod protein FlgB -2.05 1.40 1.73

flgA t1747 flagellar basal body P-ring biosynthesis protein

FlgA -1.29 1.37 1.70

sprB t2768 AraC family transcriptional regulator -3.76 1.97 4.11

sprA t2769 AraC family transcriptional regulator -3.29 1.97 3.29

- t2770 hypothetical protein -3.69 1.22 2.11

orgA t2771 oxygen-regulated invasion protein -3.90 1.34 1.79

orgA t2772 oxygen-regulated invasion protein -5.65 1.62 3.50

prgJ t2774 pathogenicity island 1 effector protein -6.05 1.43 3.83

prgI t2775 pathogenicity island 1 effector protein -6.15 1.41 3.89

prgH t2776 pathogenicity island 1 effector protein -6.36 1.57 4.02

hilA t2778 invasion protein regulator -6.98 1.54 3.67

iagB t2779 cell invasion protein -6.64 1.35 3.83

sicP t2781 chaperone -3.06 1.40 3.19

- t2782 hypothetical protein -3.10 1.56 2.98

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

37

sipF/iacP t2783 acyl carrier protein -5.62 1.46 3.43

sipA t2784 pathogenicity island 1 effector protein -5.84 1.55 3.60

sipD t2785 pathogenicity island 1 effector protein -6.24 1.48 3.87

spaS t2789 surface presentation of antigens protein SpaS -5.70 1.24 3.29

spaQ t2791 virulence-associated secretory protein -7.26 1.40 3.00

spaP t2792 surface presentation of antigens protein SpaP -6.87 1.43 3.25

spaO t2793 surface presentation of antigens protein SpaO -6.72 1.60 3.66

spaN t2794 antigen presentation protein SpaN -6.66 1.58 3.91

spaM t2795 virulence-associated secretory protein -6.91 1.76 3.83

spaL/invC t2796 ATP synthase SpaL -6.61 1.53 3.43

Spak/invB t2797 virulence-associated secretory protein -6.04 1.91 4.01

invA t2798 virulence-associated secretory protein -6.50 1.40 3.34

invE t2799 cell invasion protein -6.86 1.35 3.59

invG t2800 virulence-associated secretory protein -7.12 1.37 3.60

invF t2801 AraC family transcriptional regulator -6.97 1.27 3.84

invH t2802 cell adherance/invasion protein -4.54 1.57 2.97

sopD t2846 hypothetical protein -3.76 1.05 4.33

rtsB t4220 GerE family regulatory protein -7.59 1.99 3.58

rtsA t4221 AraC family transcriptional regulator -7.33 1.80 3.83

- t0944 lipoprotein -2.25 1.20 2.22

- t1774 hypothetical protein -2.09 1.46 2.60

lpxR t1208 hypothetical protein -7.02 1.19 3.44

srfA t1503 virulence effector protein -1.75 1.64 1.81

srfB t1504 virulence effector protein -1.48 1.58 1.88

731

Table 4. Strains and plasmids used in this study 732

Strain or plasmid Identifier Genotype or comments Source

Strains

S. Typhimurium

14028 ICC797 WT (64)

14028 ICC1765 ΔhilA:lacZ KanR This study

14028 ICC1764 ΔhilD:lacZ KanR This study

S. Typhi

Ty2 ICC1500 WT G. Dougan

Ty2 ICC1630 ΔhilA:lacZ KanR This study

Ty2 ICC1762 ΔhilD:lacZ KanR This study

Ty2 ICC1556 ΔinvA KanR (64)

Ty2 ICC1756 Δpat KanR This study

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

38

CT18 ICC1502 WT G. Dougan

129-0238 ICC1503 WT, H58 isolate G. Dougan (21)

ERL12148 ICC1504 WT, H58 isolate G. Dougan (21)

Plasmids

pKD4 pICC893 Kanamycin cassette template

plasmid

(63)

p3138 pICC2515 LacZ and kanamycin cassette

template plasmid

(41)

pKD46 pICC1298 Lambda red recombinase

plasmid

(63)

pWSK29-Spec E.V. pICC2489 Empty vector, spectinomycinR (64)

pWSK29-Spec HilD-

4HA Ty2

S. Typhi Ty2 HilD-4HA,

constitutive promoter

This study

pWSK29-Spec HilD

4HA Tm

S. Typhimurium 14028 HilD-

4HA, constitutive promoter

This study

733

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from

on January 4, 2018 by LON

DO

N S

CH

OO

L OF

HY

GIE

NE

& T

RO

PIC

AL M

ED

ICIN

Ehttp://iai.asm

.org/D

ownloaded from